Project Summary/Abstract Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) that contain thioether linkages, endowing them with unique activities and protease resistance. This proposal is focused on expanding on the already realized display of the lanthipeptide modified NisA on phage and the remarkable discovery that such phage has a radically shifted host range. Three aims are proposed to characterize, expand, and exploit this discovery. First, clear definition of the means of infection of this phage will provide guidance in engineering future variants with improved properties ? either broader host range or more focused infectivity. An array of competition binding experiments as well as mutation and truncation of NisA are proposed to investigate how the phage infects the host. Second, display of other peptides on phage will both validate the usefulness of the system to display a broad range of RiPPs and potentially further impact the infectivity of the phage. It is proposed to display both additional lanthipeptides and another class of RiPP, the lasso peptide MccJ25. Finally, clear definition of the host range achievable in the engineered phages as well as the range of genetic manipulation that can be accomplished has enormous potential to impact biotechnology through facilitating transformation of industrially relevant bacteria and to impact the academic world through increasing the speed at which knockouts of biosynthetic pathways can be constructed via CRISPR-Cas9 or homologous recombination, thereby enabling rapid determination of natural product maturation pathways or even virulence production pathways. A recent report noted that endowing native bacteria in the gut with new bacteriocin producing machinery allowed them to out-compete virulent strains, thereby restoring gut health. A robust and highly infective version of our phage may be able to perform such a transformation in situ, thereby leveraging native gut fauna to expunge virulent fauna. Moreover, such phage could use a CRISPR-Cas9 system to delete essential genes for virulence. Taken together, these aims will allow the development of this phage variant as a tool for use in biotechnology, academic, and medical fields.